The present invention relates to polynucleotide sequences which are shown herein to be associated with the regulation of angiogenesis. More specifically, the present invention relates to novel polynucleotide sequences which encode the angiogenesis inhibitor endostatin, and more particularly, the canine angiogenesis inhibitor. The invention encompasses endostatin nucleic acids, recombinant DNA molecules, cloned genes and degenerate variants thereof, vectors containing such endostatin nucleic acids, and hosts that have been genetically engineered to express and/or contain such molecules. The invention further relates to endostatin gene products and antibodies directed against such gene products. The invention further relates to methods for the identification of compounds that modulate the expression, synthesis and activity of such endostatin nucleic acids, and to methods of using compounds such as those identified herein as therapeutic agents in the treatment of angiogenesis-related disorders, including, but not limited to, cancer. The invention also relates to methods for the diagnostic evaluation, genetic testing and prognosis of an angiogenesis-related disorder, including, but not limited to, cancer.
Angiogenesis, defined as the growth or sprouting of new blood vessels from existing vessels, is a complex process that primarily occurs during embryonic development. Under normal physiological conditions in adults, angiogenesis takes place only in very restricted situations such as hair growth and wounding healing (Auerbach, W. and Auerbach, R., 1994, Pharmacol Ther 63(3):265-3 11; Ribatti et al.,1991, Haematologica 76(4):3 11-20; Risau, 1997, Nature 386(6626):67 1-4). Unregulated angiogenesis has gradually been recognized to be responsible for a wide range of disorders, including, but not limited to, cancer, cardiovascular disease, rheumatoid arthritis, psoriasis and diabetic retinopathy (Folkman, 1995, Nat Med 1(1):27-31; Isner, 1999, Circulation 99(13): 1653-5; Koch, 1998, Arthritis Rheum 41(6):951-62; Walsh, 1999, Rheumatology (Oxford) 38(2):103-12; Ware and Simons, 1997, Nat Med 3(2): 158-64). Of particular interest is the observation that angiogenesis is required by solid tumors for their growth and metastases (Folkman, 1986, Cancer Res, 46(2) 467-73. Folkman 1990, J Natl. Cancer Inst., 82(1) 4-6, Folkman, 1992, Semin Cancer Biol 3(2):65-71; Zetter, 1998, Annu Rev Med 49:407-24). A tumor usually begins as a single aberrant cell which can proliferate only to a size of a few cubic millimeters due to the distance from available capillary beds, and it can stay xe2x80x98dormantxe2x80x99 without further growth and dissemination for a long period of time. Some tumor cells then switch to the angiogenic phenotype to activate endothelial cells, which proliferate and mature into new capillary blood vessels. These newly formed blood vessels not only allow for continued growth of the primary tumor, but also for the dissemination and recolonization of metastatic tumor cells. The precise mechanisms that control the angiogenic switch is not well understood, but it is believed that neovascularization of tumor mass results from the net balance of a multitude of angiogenesis stimulators and inhibitors (Folkman, 1995, Nat Med 1(1):27-31).
One of the most potent angiogenesis inhibitors is endostatin identified by O""Reilly and Folkman (O""Reilly et al., 1997, Cell 88(2):277-85; O""Reilly et al., 1994, Cell 79(2):3 15-28). Its discovery was based on the phenomenon that certain primary tumors can inhibit the growth of distant metastases. O""Reilly and Folkman hypothesized that a primary tumor initiates angiogenesis by generating angiogenic stimulators in excess of inhibitors. However, angiogenic inhibitors, by virtue of their longer half life in the circulation, reach the site of a secondary tumor in excess of the stimulators. The net result is the growth of primary tumor and inhibition of secondary tumor. Endostatin is one of a growing list of such angiogenesis inhibitors produced by primary tumors. It is a proteolytic fragment of a larger protein: endostatin is a 20 kDa fragment of collagen XVIII (amino acid H1132-K1315 in murine collagen XVIII). Endostatin has been shown to specifically inhibit endothelial cell proliferation in vitro and block angiogenesis in vivo. More importantly, administration of endostatin to tumor-bearing mice leads to significant tumor regression, and no toxicity or drug resistance has been observed even after multiple treatment cycles (Boehm et al., 1997, Nature 390(6658):404-407). The fact that endostatin targets genetically stable endothelial cells and inhibits a variety of solid tumors makes it a very attractive candidate for anticancer therapy (Fidler and Ellis, 1994, Cell 79(2):185-8; Gastl et al., 1997, Oncology 54(3):177-84; Hinsbergh et al., 1999, Ann Oncol 10 Suppl 4:60-3). In addition, angiogenesis inhibitors have been shown to be more effective when combined with radiation and chemotherapeutic agents (Klement, 2000, J. Clin Invest, 105(8) R15-24. Browder, 2000, Cancer Res. 6-(7) 1878-86, Arap et al., 1998, Science 279(5349):377-80; Mauceri et al., 1998, Nature 394(6690):287-91).
Cancer is not only devastating to humans, but is also the most common cause of natural death in dogs. (Bronson, 1982, Am J Vet Res, 43(11) 2057-9). Dogs develop tumors twice as frequently as humans and it has been reported that 45-50% of dogs that live to 10 years or older die of cancer; regardless of age, and that 23% of dogs that present for necropsy died of cancer(Bronson, 1982, Am J Vet Res, 43(11) 2057-9). Surgical removal of the tumor is the most common treatment, but the prognosis for invasive/metastatic tumor is very poor, with median survival time ranging from weeks to months. Other treatments, such as radiation therapy and chemotherapy, have only very limited success (Bostock, 1986, Br Vet J 142(6):506-15; Bostock, 1986, Br Vet J 142(1):1-19; MacEwen, 1990, Cancer Metastasis Rev 9(2): 125-36). Thus, more effective treatments for angiogenic diseases, such as, for example, canine cancers, are necessary.
The present invention encompasses novel nucleotide sequences that are associated with angiogenesis related disorders, e.g., cancer. The invention more specifically relates to nucleotide sequences that encode endostatin. In addition, endostatin nucleic acids, recombinant DNA molecules, cloned genes or degenerate variants thereof are provided herein. The invention also provides vectors, including expression vectors, containing endostatin nucleic acid molecules, and hosts that have been genetically engineered to express and/or contain such endostatin gene products.
The invention further relates to novel endostatin gene products and to antibodies directed against such gene products, or variants or fragments thereof.
The invention further relates to methods for modulation of endostatin-mediated processes and for the treatment of disorders involving angiogenesis, such as cancer, including the amelioration or prevention of at least one symptom of the disorders, wherein such methods comprise administering a compound which modulates the expression of an endostatin gene and/or the synthesis or activity of an endostatin gene product. In one embodiment, the invention relates to methods for the use of a novel endostatin gene product or fragment, analog, or mimetic thereof, or an antibody or antibody fragment directed against an endostatin gene product, to treat or ameliorate a symptom of such disorders.
Such disorders include, but are not limited to, angiogenesis-dependent cancer, including, for example, solid tumors, blood born tumors such as leukemias, and tumor metastases; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; wound granulation; corornary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; diabetic neovascularization; macular degeneration; fractures; vasculogenesis; hematopoiesis; ovulation; menstruation; and placentation.
The invention further relates to methods for modulation of endostatin-mediated processes and for the treatment of disorders involving abnormal stimulation of endothelial cells, including the amelioration or prevention of at least one symptom of the disorders, wherein such methods comprise administering a compound which modulates the expression of an endostatin gene and/or the synthesis or activity of an endostatin gene product. In one embodiment, the invention relates to methods for the use of a novel endostatin gene product or fragment, analog, or mimetic thereof, or an antibody or antibody fragment directed against an endostatin gene product, to treat or ameliorate a symptom of such disorders.
The endothelial cell proliferation inhibiting proteins of the present invention are useful in the treatment of disease of excessive or abnormal stimulation of endothelial cells. These diseases include, but are not limited to, intestinal adhesions, atherosclerosis, scleroderma, and hypertrophic scars, i.e., keloids. They are also useful in the treatment of diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa) and ulcers (Helobacter pylori).
The invention further relates to methods for blocking interactions between endostatin and its respective receptors with analogs that act as receptor antagonists. These antagonists may promote endothelialization and vascularization. Such effects may be desirable in situations including, but not limited to, inadequate vascularization of the uterine endometrium and associated infertilty, wound repair, healing of cuts and incisions, treatment of vascular problems in diabetics, especially retinal and peripheral vessels, promotion of vascularization in transplanted tissue including muscle and skin, promotion of vascularization of cardiac muscle especially following transplantation of a heart or heart tissue and after bypass surgery, promotion of vascularization of solid and relatively avascular tumors for enhanced cytotoxin delivery, and enhancement of blood flow to the nervous system, including but not limited to the cerebral cortex and spinal cord.
The term xe2x80x9cendostatin-related disorderxe2x80x9d as used herein, refers to disorders involving an endostatin gene or gene product, or an aberrant level of endostatin gene expression, gene product synthesis and/or gene product activity, respectively, relative to levels found in normal, unaffected, unimpaired individuals, levels found in clinically normal individuals, and/or levels found in a population whose levels represent baseline, average endostatin levels.
The term xe2x80x9cendostatin-mediated processxe2x80x9d as used herein, includes processes dependent and/or responsive, either directly or indirectly, to the level of expression, gene product synthesis and/or gene product activity of endostatin genes.
In another embodiment, such methods can comprise modulating the level of expression or the activity of an endostatin gene product in a cell such that the endostatin-mediated process or the disorder is treated, e.g., a symptom is ameliorated. In another embodiment, such methods can comprise supplying a nucleic acid molecule encoding an endostatin gene product to increase the level, expression or activity of the endostatin gene product within the cell such that the endostatin-mediated process or the disorder is treated, e.g., a symptom is ameliorated. The nucleic acid molecule encoding the endostatin gene product can encode a mutant endostatin gene product with increased activity or expression levels.
The invention still further relates to methods for modulation of endostatin-mediated processes or the treatment of endostatin-related disorders, such as cancer, including, but not limited to, disorders resulting from endostatin gene mutations, and/or an abnormal levels of endostatin expression or activity and disorders involving one or more endostatin genes or gene products, wherein treatment includes the amelioration or prevention of at least one symptom of such disorders. In one embodiment, such methods can comprise supplying a mammal in need of treatment with a nucleic acid molecule encoding an unimpaired endostatin gene product such that the unimpaired endostatin gene product is expressed and the disorder is treated, e.g., a symptom is ameliorated. In another embodiment, such methods can comprise supplying a mammal in need of treatment with a cell comprising a nucleic acid molecule that encodes an unimpaired endostatin gene product such that the cell expresses the unimpaired endostatin gene product and the disorder is treated, e.g., a symptom is ameliorated. In yet another embodiment, such methods comprise supplying a mammal in need of treatment with a modulatory compound, such as, for example, a small molecule, peptide or antibody that is capable of modulating the activity of an endostatin gene or gene product.
In addition, the present invention is directed to methods that utilize endostatin gene sequences and/or endostatin gene product sequences for the diagnostic evaluation, genetic testing and/or prognosis of angiogenesis-related disorders, such as cancer. For example, the invention relates to methods for diagnosing angiogenesis-related disorders, e.g., cancer, wherein such methods can comprise measuring endostatin gene expression in a patient sample, or detecting an endostatin mutation that correlates with the presence or development of such a disorder, in the genome of a mammal suspected of exhibiting such a disorder.
The present invention also is directed to utilizing the endostatin gene sequences and/or gene products as markers for mapping of the human chromosome.
The invention still further relates to methods for identifying compounds capable of modulating the expression of an endostatin gene and/or the synthesis or activity of an endostatin gene product, wherein such methods comprise contacting a compound with a cell that expresses such an endostatin gene, measuring the levels of endostatin gene expression, gene product expression or gene product activity, and comparing such levels to the levels of endostatin gene expression, gene product, or gene product activity produced by the cell in the absence of such compound, such that if the level obtained in the presence of the compound differs from that obtained in its absence, a compound capable of modulating the expression of the endostatin gene and/or the synthesis or activity of the endostatin gene product has been identified.
As used herein, the following terms shall have the abbreviations indicated.